We have discovered a series of heme proteins in the saliva of the blood-sucking bug Rhodnius prolixus that act as vasodilators, anti-platelet aggregation and anti-histaminic agents when injected into the victim. These heme proteins are novel Fe(lll) proteins that are reversible carriers of nitric oxide (NO) and tight binders of histamine. Because of their NO-carrying function, these proteins have been named "nitrophorins," and we have characterized them in some detail during the first and second grant periods, including structural, infrared, resonance Raman, NMR, EPR and Mossbauer spectroscopies and electrochemical and kinetics investigations. We have also partially characterized a nitrophorin from the saliva of the bedbug, Cimex lectularius. Our goal is to continue to extensively characterize them, in terms of their structures, physical properties and chemical and biological reactivity, in order to achieve a better understanding of the interaction of NO and histamine with heme proteins; to provide insight concerning other NO-binding heme proteins and the general factors that mediate the reactivity of coordinated NO in biological systems; and to provide perspective on the biochemical, physiological, and pharmacological roles that these interactions may play in human health. Thus, we are not only interested in the specific properties of these nitrophorins, but in how they can help us to unlock many of the secrets involving the role of nitric oxide and histamine in biology and human health. The goals of the present grant proposal involve all four of the proteins from Rhodnius prolixus, NP1-NP4, and a number of carefully-selected sitedirected mutants of these proteins, as well as detailed characterization of the nitrophorin from Cimex lectularius and its site-directed mutants. Our specific aims are: 1. To investigate additional properties of the distal binding site of the heme by preparing and studying the spectroscopic and electrochemical properties of carefully-chosen site-directed mutants of the nitrophorins of R. prolixus. 2. To investigate the factors which determine the reduction potentials of the heme of R. prolixus by preparing and studying the electrochemistry of mutants of charged residues near the heme, and to determine which residues could be modified to alter the function of the nitrophorins. 3. To determine the pKas of the charged residues near the heme by preparing the glutamate and aspartate isotopically-labeled proteins utilizing auxotrophic bacterial strains, and then carrying out pH titrations with 13C detection of the carboxylate and beta-CH2 protons. 4. To continue determination of the 3-dimensional structure of NP2 by NMR spectroscopy, both as the apoprotein and as two holoprotein complexes, the NP2-imidazole and the reduced NP2-CO complex. The results obtained will be used to investigate the dynamics of the beta-barrel protein in each of these forms. 5. To continue investigations of the Mossbauer spectra of the nitrophorin-NO complexes in both the Fe(III)-NO and Fe(II)-NO forms, and to carry out detailed DFT calculations aimed at shedding light on the electron configuration and bonding in these complexes. 6. To investigate the nitrophorin from Cimex lectularius in collaboration with the Montfort group by studying the NMR, EPR and Mossbauer spectra, spectroelectrochemistry, and ligand binding properties of the wild type protein and a number of well-chosen site-directed mutants of it in order to better understand the mechanism of action of this protein, and how its binding of two NO molecules may relate to the reactivity of NO with other heme-thiolate proteins.